Picture Blog: mRNA Reprogramming for Human iPSCs without B18R!

Human induced pluripotent stem cells provide a great route towards personalized medicine and high accuracy drug screening. Allele Biotech has developed the most efficient method of making human iPSCs by using enhanced mRNAs, which have been adopted by leading pharmaceutical companies for clinical trials. The effects from medium-supplementing mRNAs are robust yet transient, and highly specific compared to both miRNAs (off-targets) and small molecules (unknown targets). To repress cellular immune response to introduced RNA molecules, viral protein B18R was previously used during mRNA reprogramming.

B18R is relatively expensive and inconvenient to use because it requires pre-aliquoting and -80C storage. The protocol has recently been dramatically improved at Allele through an NIDA-funded project. In our latest reprogramming run, all we needed to do was to include mRNA complex in the supplement during medium change for just a week without the need of adding any other type of molecules (such as B18R, miRNA, or chemicals) to help the mRNA mix, unlike all other known mRNA-reprogramming protocols. This advancement can make reprogramming human fibroblasts to footprint-free and xeno-free iPSCs a routine experiment for any lab to perform.

Tags: apaptosis, B18R, cellular immune response, footprint-free iPSC, iPSCs, miRNA reprogramming, mRNA iPSC, R-iPSCs, reprogramming, small-molecule compounds

mRNA Transfection for Better Transgene Expression

Different approaches have been developed to over-express or ectopically express a protein in cells: peptide or full length recombinant protein transfer, viral gene transfer, non-viral DNA transfer and non-viral mRNA transfer.

1) Peptide transfection can be efficient, yet it is limited to only a small part of the protein, limiting the functional potential. Protein transfection is not consistent enough so far, because of the complicated properties of different proteins. Allele Biotech has tested dozens of proteins with several proprietary reagents, leader peptides, etc. but we have decided not to carry a protein transfection product line due to its instability. Furthermore, protein production is an expensive and laborious process.

2) Viral gene transfer is very effective, such as the HIV-based lentivirus or MMLV-based retrovirus, adenovirus, adeno-like virus or baculovirus, etc. However, the potent side-effect will still need to be considered for certain applications, especially involving clinical studies. Nevertheless, as research tools, viral gene transfer is still a highly preferred method. Allele Biotech has been providing the most effective platform for both MMLV-based and HIV-1-based retrovirus packaging. Check out our product website for details.

3) Non-viral DNA transfer is the most widely used transgene method in the biological research community, due to the simplicity of the procedure. There are many commercial kits on the market. However, the low efficiency for transfecting most primary cells significantly limits their use. In recent years, several leading biotech companies have developed various electroporation systems to improve the transfection efficiency and cell viability; although these improvements help with getting DNA inside the cytoplasm, they hardly help transport it into nucleus where DNA is transcribed.

4) Non-viral mRNA transfer has been around for a long time, but it is not widely used. It made a big splash recently through its use for iPSCs reprogramming. IPSCs factor mRNAs greatly improved the iPSCs induction efficiency and completely avoided the viral integration. Other well-known examples of mRNA transfection include loading special cancer antigens or HIV antigens to dendritic cells (DCs) in vitro for personal immunotherapy. PSA antigen expressing DCs transfected by mRNA has moved on to Phrase I Clinical Trials for this purpose.

New Product of the Week: 3C protease immobilized on beads for GST, His tag removal, email oligo@allelebiotech.com for details.

Promotion of the week: 10% off on all fluorescent proteins. To redeem, email oligo@allelebiotech.com along with PROMO code: JELLYFISH

Tags: 5-methylcytidine (5mC), B18R, in vitro transcription, interferon, iPSC factor mRNA, iPSC Warren, modified mRNA, mRNA iPSC, mRNA transfection, mRNA transfer, mRNA transgene, pseudouridine (psi), RiPSC, T7 RNA polymerase, VPA